Liposome-encapsulated glutathione for oral administration

ABSTRACT

The invention is a composition administrable orally to provide systemic glutathione (reduced) and a method for providing systemic glutathione by oral administration of glutathione (reduced) in a liposome encapsulation. The administration of a therapeutically effective amount of oral liposomal glutathione (reduced) results in improvement of symptoms in disease states related to glutathione deficiency such as Parkinson&#39;s disease and cystic fibrosis. Compounds enhancing the effect of the liposomal glutathione are contemplated such as Selenium, EDTA, carbidopa, and levodopa.

CONTINUATION DATA

This application claims priority from co-pending application U.S. Ser.No. 11/163,979 filed Nov. 6, 2005, which in turn claims priority from ofU.S. Provisional Application No. 60/597,041 both entitled LiposomalFormulation for Oral Administration of Glutathione (Reduced), and is acontinuation application of Ser. No. 11/163,979. Although thisapplication claims the priorities in Ser. No. 11/163,979, including toU.S. Provisional patent application 60/522,785 filed on Nov. 7, 2004 forpurposes of further continuing applications and the like, no claim ismade to such priority for the claim of this patent application.

SUMMARY OF INVENTION

The invention is a composition of glutathione (reduced) in a liposomeconstructed to stabilize the glutathione in a physiologically activestate which can be orally administered and delivers a therapeuticallyeffective amount of glutathione (reduced) to improve symptoms in diseasestates by transfer of the glutathione into the cells of the body, andmethod of manufacture of the same. Previous art did not enable oraladministration of glutathione (reduced) in a therapeutically effectiveway. The invention is also a method of encapsulating glutathione(reduced) in a liposome constructed to stabilize the glutathione in aphysiologically active state and to enable the oral administration of atherapeutically

effective amount of glutathione (reduced) to improve symptoms in diseasestates by transfer of the glutathione into the cells of the body.Compounds enhancing the effect of the liposomal glutathione arecontemplated such as Selenium, EDTA, carbidopa and levodopa.

TECHNICAL FIELD

The invention relates to the field of delivery of a nutrient substance,glutathione in the biochemically-reduced form, in a liposomalpreparation that allows the novel delivery mode of oral delivery ofglutathione (reduced) in a sufficient amount to improve the condition ofa disease state related to glutathione deficiency. The delivery may alsobe accomplished via absorption across the mucosa of the nose, mouth,gastrointestinal tract, after topical application for transdermal, orintravenous infusion.

BACKGROUND

The tripeptide L-glutathione (GSH) (gamma-glutamyl-cysteinyl-glycine) iswell known in biological and medical studies to serve several essentialfunctions in the cells of higher organisms such as mammals. It isfunctional when it appears in the biochemical form known as the reducedstate (GSH). When oxidized, it forms into a form known as a dimer(GSSG).

Glutathione in the reduced state (GSH) functions as an antioxidant,protecting cells against free-radical mediated damage, a detoxifyingagent by transporting toxins out of cells and out of the liver, and acell signal, particularly in the immune system.

A deficiency of glutathione (reduced) may lead to damage to cells andtissues through several mechanisms including the accumulation of anexcess of free radicals which causes disruption of molecules, especiallylipids causing lipid peroxidation, and which combined with toxinaccumulation will lead to cell death. These mechanisms are oftenreferred to as oxidation stress as general term. The lack of sufficientglutathione in the reduced state relative to the oxidized state may bedue to lack of production of glutathione (reduced) or an excess of thematerials such as toxins that consume glutathione (reduced). The lack ofglutathione (reduced) may manifest as a systemic deficiency or locallyin specific cells undergoing oxidation stress.

Deficiency of glutathione in the reduced state contributes to oxidativestress, which plays a key role in aging and the pathogenesis of manydiseases such as

Cystic fibrosis

Liver disease

Parkinson's disease

Alzheimer's disease

Heart attack and Stroke

Diabetes

Viral disease

Free radical damage from nuclear, biological or chemical insult

Free radical damage from bacterial infection

Immune system modulation after vaccination

The use of the term “glutathione” or “glutathione (reduced)” will referto glutathione in the reduced state.

Replacing glutathione in deficient states has been difficult because ofthe lack of direct absorption of glutathione after oral administration.Glutathione is a water-soluble peptide. This characteristic ofglutathione is thought to prevent its absorption into the system afteroral ingestion of glutathione. The fate of direct oral ingestion ofglutathione has been demonstrated in a clinical study showing that 3grams of glutathione delivered by oral ingestion does not elevate plasmaglutathione levels.

Building glutathione level in the body has required the use of eitherdirect intravenous infusion of glutathione or the administration ofbuilding blocks of glutathione such as cysteine (Smith et al, U.S. Pat.No. 6,495,170) as the direct oral administration has been documented toneither elevate glutathione levels (Rowe et al, U.S. Pat. No. 5,747,459)nor have clinical benefit.

The intravenous administration of glutathione has been reported to havebenefit in improving blood flow in peripheral vascular disease andimproving symptoms related to Parkinson's disease. This applicationincludes claims for the use of the invention in its oral form in thetreatment of Parkinson's disease, Cystic Fibrosis, vascular disease,diabetes, as well as inflammatory diseases of the respiratory tract suchas chronic sinus disease, emphysema and allergy. A particular advantageof this invention is the ability of liposomal encapsulation ofglutathione (reduced) is to deliver the reduced glutathione to theintracellular compartment of cells, such as, but not limited to, redblood cells. This characteristic of the invention is important inindividuals with defects in the transport of glutathione into cells suchas the defect seen in cystic fibrosis.

The clinically effective use of glutathione in its pure form directlywithout any additive encapsulation or transformation (in the “neat”form) has been limited to the intravenous administration of thebiochemical in the reduced state. As glutathione is unstable in solutionwithout the protection from oxidation offered by this invention, thereare limitations to the stability of solution preparations. Demopoulus etal, U.S. Pat. No. 6,204,248, describe a method of preparation ofglutathione in combination with crystalline ascorbic acid enclosed in agel cap for oral administration. Demopoulus et al, U.S. Pat. No.6,204,248 describes the use of the glutathione in combination withcrystalline ascorbic acid enclosed in a gel cap for oral administrationto alter redox state of cells and improve disease processes. Demopouluset al, U.S. Pat. No. 6,350,467 references the use of the glutathione incombination with crystalline ascorbic acid enclosed in a gel cap fororal administration to treat additional disease states. A recent patent,Smith, U.S. Pat. No. 6,764,693 references the use of liposomescontaining a combination of glutathione in combination with at least oneother antioxidant material to increase intracellular and extra cellularantioxidants. There is no claim for the use of liposomal glutathioneeither individually or in combination with other antioxidants for thetreatment of Parkinson's disease or Cystic Fibrosis. Additionally theclaim for activity of the liposome in Smith '693 is a population ofliposomes suitable for undergoing peroxidation and lysis, releasingtheir contents into the circulation. The preferred method of compositionof the liposome claimed in this invention is for a liposome thatfunctions by fusion and transfer of the glutathione content into cells.Evidence for this method of action is provided in the clinical examplesof improvement in the red blood cell level of glutathione parallelingclinical improvement in individuals with Cystic Fibrosis.

A liposome is a microscopic fluid filled pouch whose walls are made ofone or more layers of phospholipid materials identical to thephospholipid that make up cell membranes. Lipids can be used to delivermaterials such as drugs to the body because of the enhanced absorptionof the liposome. The outer wall of the liposome is fat soluble, whilethe inside is water-soluble. This combination allows the liposome tobecome an excellent method for delivery of water-soluble materials thatwould otherwise not be absorbed into the body. A common material used inthe formation of liposomes is phosphatidylcholine, the material found inlecithin. A more detailed description of the constituents of thisinvention is provided.

Cystic Fibrosis (CF) is an inherited disorder that affects approximately30,000 children in the United States. It is the most common geneticdisorder and the largest genetic killer of children. Cystic fibrosis ischaracterized by the production of thick mucus in the lungs and sinusesand leads to recurring infections as well as gastrointestinaldysfunction. At the present time there is no cure and none of thetherapies offered correct the underlying cellular defect. The currenttherapy is oriented toward strategies for removing mucus with physicaltherapies and antibiotics therapy for treating the infections thatinevitably occur. Even with intensive therapies the current median ageof survival of people with CF is the early 30's. Although CF has seriousclinical implications for the gastrointestinal and genital tracts,pulmonary disease is the primary cause of death in 90% of CF patients.

Recent research demonstrates that the gene defect in CF codes for aprotein that carries materials across cell membranes. This is associatedwith inability of people with CF to carry chloride into cells, whichresults in the characteristic of the accumulation of an excess ofchlorides on the skin. This observation leads to the initial testcharacteristic of CF, the sweat chloride test. The protein that carrieschloride, the CF transmembrane conductance regulator (CFTR) protein, hasalso been found to carry other large anions such as glutathione acrosscell membranes. The lung epithelial lining fluid of adults with CF haslower glutathione levels than controls and laboratory animal studies ofmice without the CFTR protein gene confirm the observation thatglutathione transport is deficient in CF. In addition, the ratio ofreduced to oxidized GSH in CF individuals is abnormal with an excess ofoxidized glutathione present. The lack of glutathione transport has ledto the observation that oral supplements used to increase glutathionelevels may not have the effect of reaching the interior glutathionedeficient cells, such as red blood cells (RBC). Thus, the RBCglutathione level has been proposed as a potential marker of diseaseseverity in individuals with cystic fibrosis.

The majority of glutathione is formed in the liver and released into theblood. It appears that the membrane transport defect in cystic fibrosisaffects the ability of the red blood cell to maintain adequate levels ofglutathione in side of cells such as red blood cells. Thus, more severecases of cystic fibrosis are associated with a decrease in the red bloodcell level of glutathione that exceeds that found in the plasma. Theaddition of the presently described invention, liposomal glutathione, tothe system can raise the level of glutathione inside the cells of thebody such as the red blood cells.

Liposomes have been documented to fuse with red blood cells and delivertheir content into the cells (Constantinescu I, Artificial cells, bloodsubstitutes, and immobilization biotechnology. 2003 November;31(4):395-424).

The clinical examples demonstrate that the present invention can raisethe glutathione level of red blood cells of individuals with CysticFibrosis. As these individual's cells have a genetic defect in thetransport of glutathione across cell membranes the increase observed inthe red blood cell levels of glutathione is demonstrated to occur afteroral ingestion of the invention and by a mechanism such as fusion of theliposome. Release of the glutathione reduced into the systemiccirculation would not result in the elevation of glutathione seen in theindividuals with Cystic Fibrosis.

Liposomes are able to convey their contents to cells by one of fourmethods:

Adsorption: The wall of the liposome becomes adherent to the cell andreleases the content of the liposome into the cell.

Endocytosis: In endocytosis the cell engulfs the liposome creating anadditional lamella around the liposome, which is dissolved inside thecell, releasing the contents of the liposome. In the process ofendocytosis a portion of the plasma membrane is invaginated and pinchedoff forming a membrane-bounded vesicle called an endosome.

Lipid exchange: The lipid contents of the liposome and the cell exchangetheir lipid contents, releasing the contents of the liposome

Fusion: The melding or the liposome membrane with the membrane of thecell, carrying their contents of the liposome into the cell.

One or more of these mechanisms is at play in the described invention,allowing delivery of glutathione into the cells of individuals withcystic fibrosis.

While tableted or other solid forms of administration of nutrients isconvenient for many individuals there is a significant segment of thepopulation for whom swallowing a tablet is not possible. This can be dueto age, such as the pediatric segment of the population or the other endof the age spectrum, the geriatric population, many of whom find pillswallowing difficult. For this reason, as well as ease of dosecalculation, liquid gel delivery of glutathione will be more universallyacceptable. Another advantage is that the present invention enablesadministration of a larger quantity of GSH in a single dose than otherforms of non-parenteral administration as well as enabling incrementaladjustment of doses for children and adults.

Liposome delivery of glutathione as described in this invention isparticularly efficient for providing glutathione across cell membranes,which is critical for the management of Cystic Fibrosis. This disease isa genetic deficiency of the ability to transport certain molecules likeglutathione across cell membranes resulting in an intracellulardeficiency of glutathione. The difficulties associated with CysticFibrosis occur in the early stages of life, a time in which theingestion of liquids is the only option for the individuals due to theiryoung age.

Parkinson's Disease

Parkinson's disease (PD) is a medical condition associated with theneuro-motor system and characterized by four primary symptoms:

Tremor or trembling in hands, arms, legs, jaw, and face

Rigidity or stiffness of the limbs and trunk

Bradykinesia, or slowness of movement

Postural instability or impaired balance and coordination.

Individuals with Parkinson's disease may have difficulty walking,talking, or completing other simple tasks. The disease is both chronicand progressive. Early symptoms are subtle and occur gradually and oftenprogress.

The brain is the body's communication headquarters. It is thecoordinator of information received from the various parts of thesensory system. The brain processes the information in an organizedfashion and relays the information to the motor system for movement.This highly organized passage of information can become disrupted withthe slightest offset of the assembly-line fashion of the cellularchemical sequence resulting in major abnormalities.

There are two areas of the brain that are specifically related to motormalfunctions, the substantia nigra and the striatum. The substantianigra is located in the midbrain, halfway between the cerebral cortexand the spinal cord. In healthy people, the substantia nigra containscertain nerve cells, called nigral cells that produce the chemicaldopamine. Dopamine travels along nerve cell pathways from the substantianigra to another region of the brain, called the striatum. In thestriatum, dopamine activates nerve cells that coordinate normal muscleactivity.

In people with Parkinson disease, nigral cells deteriorate and die at anaccelerated rate, and the loss of these cells reduces the supply ofdopamine to the striatum. Dopamine is one of the chemical messengersresponsible for transmitting signals in the brain and must be balancedwith other neurotransmitters such as acetylcholine. Without adequatedopamine, nerve cells of the striatum activate improperly, impairing aperson's ability to control muscular functions such as walking, balance,and muscular movement.

It appears that the substancia nigra cells may be particularlyvulnerable to oxidation stress. Oxidation stress occurs in thesubstancia nigra cells because the metabolism of dopamine requiresoxidation and can lead to the formation of free radicals from hydrogenperoxide formation. In the presence of metal ions such as iron thehydrogen peroxide can form hydroxyl ions, which can be very damaging tocells. The hydrogen peroxide is normally detoxified by reducedglutathione in the reaction catalyzed by Glutathione peroxidase, thus anincreased rate of dopamine turnover or a deficiency of glutathione couldlead to oxidative stress. Thus, it appears that free radicals may be oneof the important agents responsible for destruction of substantia nigraneurons, leading to Parkinson's disease. While Parkinson's disease hasbeen treated with some success with intravenous infusion of glutathione,there has been no reported success in the use of oral glutathione in thetreatment of Parkinson's disease. Sechi G, Deledda M G, Bua G, Satta WM, Deiana G A, Pes G M, Rosati G. Reduced intravenous glutathione in thetreatment of early Parkinson's disease, Prog Neuropsychopharmacol BiolPsychiatry, 1996 October; 20(7):1159-70 PMID: 8938817.

The clinical improvement from this invention seen in patients withParkinson's disease suggests that oral liposomal glutathione is not onlyavailable systemically it also is absorbed into the central nervoussystem.

Several studies have demonstrated a deficiency of the antioxidantbiochemical reduced glutathione in substantia nigra cells of individualswith Parkinson's disease. The magnitude of the reduction in glutathioneseems to parallel the severity of the disease.

The standard treatment of Parkinson's disease has relied on thereplacement of dopamine in a form called levodopa. Levodopa, also knownas L-Dopa (from the full name L-3,4-dihydroxyphenylalanine) is a neutralamino acid found naturally in plants and animals and is used to treatthe stiffness, tremors, spasms, and poor muscle control of Parkinson'sdisease. After oral ingestion, levodopa is absorbed through the smallintestine. Levodopa's structure enables it to enter the brain, wherenerve cells can decarboxylate the levodopa and create dopamine toreplenish the brain's dwindling supply. Dopamine cannot be givendirectly because it doesn't cross the blood-brain barrier, the elaboratemeshwork of fine blood vessels and cells that filters blood reaching thebrain. Levodopa crosses the blood-brain barrier by way of the largeneutral amino acid carrier transport system. When levodopa is takenalone, however, about 95% of it is metabolized to dopamine in the body,before it ever reaches the brain. Instead of being used by the brain thedopamine circulating through the body can produce side effects such asnausea or vomiting before it is broken down in the liver.

To limit side effects, levodopa is usually given in combination withcarbidopa to increase the availability and utilization of levodopa.Carbidopa inhibits peripheral decarboxylation of levodopa but notcentral decarboxylation because it does not cross the blood-brainbarrier. Since peripheral decarboxylation is inhibited, this allows morelevodopa to be available for transport to the brain, where it will beconverted to dopamine, and relieve the symptoms of Parkinson's.Carbidopa and levodopa are given together in medications with the tradenames Stalevo (a registered trademark of Orion Pharma Inc.) or Sinemet(a registered trademark of Bristol-Myers-Squibb) and in various genericforms. The addition of carbodopa is so effective that the dose oflevodopa must be reduced by 80% when the two are use together. Thisdecreases the incidence of levodopa-induced side effects. When givenwith carbodopa the half-life of levodopa increases from 1 hr to 2 hr(may be as high as 15 hr in some clients). About 30% of the carbidopa isexcreted unchanged in the urine.

Also being used for PD are some dopamine agonists such as pramipexoledihydrochloride sold under the name MIRAPEX (a registered trademark forco-marketing by Boehringer Ingelheim Pharmaceuticals, Inc. and Pfizer,Inc.), which can also be used with the oral liposomal reducedglutathione.

Carbidopa/levodopa lessens the rigidity and slow movement associatedwith Parkinson's disease, but is less effective in treating tremor orbalance problems.

A dilemma that has been noted in recent studies is that theadministration of dopamine (singly or in the combined form of carbidopaand levodopa) results in an increase in the formation of free radicalsand the continuation of the disease process. Thus, while theadministration of levodopa offers amelioration of the symptoms ofParkinson's disease it does not change the underlying mechanisms of freeradical formation, oxidation and loss of glutathione intracellular.After several years of use the effectiveness of carbidopa/levodopadecreases and patients need higher and more frequent doses to controltheir symptoms.

Several studies have demonstrated a deficiency in the antioxidantbiochemical called reduced glutathione in specific brain cellsassociated with movement disorders called the substantia nigra (Pearce RK, Owen A, Daniel S, Jenner P, Marsden C D. Alterations in thedistribution of glutathione in the substantia nigra in Parkinson'sdisease. Journal of Neural Transmission, 1997:104(6-7):661-77. PMID:9444566).

The magnitude of the decrease of available, functional reducedglutathione inside specific brain cells seems to parallel the severityof the disease. It is theorized that the sequence of events in creatingthe dopamine loss found in Parkinson's disease involves a more rapidturnover of dopamine in the substancia nigra cells due to an increase inthe formation of hydrogen peroxide. The presence of this free radicalforming material is apparently associated with either a lack of reducedglutathione or an accumulation of the glutathione in the oxidized state.A publication from Italy in 1996 explored the possibility oftherapeutically supporting the glutathione deficient cells with the useof intravenous glutathione. Glutathione was administered intravenouslyto 9 patients, in the dose of 600 mg twice a day, for 30 days. Patientswere treated with the standard therapy using carbodopa-levodopa. It wasnoted that all patients improved significantly after glutathionetherapy, with a 42% decline in disability. The therapeutic benefitlasted for 2-4 months. The authors noted that glutathione hassymptomatic efficacy and speculated that glutathione could possiblyretard the progression of the disease. No reference was made to reducedglutathione in a liposome (Sechi G, Deledda M G, Bua G, Satta W M,Deiana G A, Pes G M, Rosati G. Reduced intravenous glutathione in thetreatment of early Parkinson's disease. Progress inNeuro-psychopharmacology & Biological Psychiatry, 1996 October;20(7):1159-70. PMID: 8938817).

The problem that this invention solves is to restore the reducedglutathione level in the brain cells associated with Parkinson's diseasein a way that can be effectively utilized by patients, and presumablythereby alter the metabolism of dopamine toward normal, allowing anormal response to dopamine. At present no patent claims and noliterature suggests the use of liposomal glutathione for the treatmentof Parkinson's disease.

A preferred mode of the invention involves administration of sufficientglutathione, reduced, orally via the liposomal glutathione to restoremetabolism of dopamine to a functional state. This allows for animprovement in symptoms by allowing the dopamine responsivenigro-striatal cells to respond to lower amounts of available dopamine.When introduced to an individual with Parkinson's disease who is takinglevodopa/carbodopa it allows the individual to respond either moreefficiently to their current dosing schedule, allowing a lower doseplateau than they would have otherwise. In addition the application ofthe invention will allow some individuals to respond to lower doses oflevodopa/carbodopa. For individuals displaying early symptoms ofParkinson's disease the application of the invention may allow them todelay the need for the administration of levodopa/carbodopa.

Individuals with advancing Parkinson's disease may develop a variety ofmotor complications associated with levodopa therapy. Fluctuations inmotor functions, such as early morning akinesia and “wearing off” areindications for the use of the liposomal glutathione invention.

The invention is a combination for administering a therapeuticallyeffective amount of glutathione orally to an individual with Parkinson'sdisease, using a liposomal encapsulation of glutathione. Prior to theintroduction of this invention, the only method for administering atherapeutically effective amount of glutathione was the intravenousinfusion of glutathione. While intravenous infusion is useful forestablishing the therapeutic efficacy of glutathione in an individual,the costs and inconvenience of intravenous administration made itextremely difficult to administer repeated doses for continued therapy.The invention is also useful in the same way for the more general classof neurodegenerative diseases like Parkinson's disease.

While there have been reports of intravenous liposome uptake protectingliver cells in animals exposed to material toxic to the liver (WendelA., Hepatic lipid peroxidation: caused by acute drug intoxication,prevented by liposomal glutathione, International Journal ClinicalPharmacology Research, 1983; 3(6):443-7. PMID: 6678834), there are noprevious reports of benefit of oral liposomal glutathione in thetreatment of human disease processes nor art claiming the use of oralliposomal glutathione in the treatment of disease states such asParkinson's disease and Cystic Fibrosis.

Because of its lack of systemic availability from oral administration,glutathione has not been used in an oral form for the treatment ofdisease states. This invention creates a composition incorporatingglutathione which is effective upon administration orally, a method ofmanufacture of a composition incorporating glutathione which iseffective upon administration orally, and is a method of administeringreduced glutathione orally, by incorporating the reduced glutathioneinto a liposome, which increases the absorption of glutathione both fromthe gastrointestinal tract and into the cells of the body. In addition,the use of the liposome encapsulation prevents or slows the degradationof the active form of glutathione in the reduced state from progressingto the oxidized state before systemic uptake. The method ofadministration thus improves bio-availability of the glutathione both byabsorption, and also by maintaining the active antioxidant state of thereduced glutathione.

The liposome preparations claimed in this invention allows themanufacture of a stable product, which can be used for theadministration of glutathione in a form that is convenient. Theliposome-glutathione preparation described is also stable fromoxidation, allowing a two year, unrefrigerated shelf-life of theproduct, and has specific characteristics of uptake into cell membranesthat improve its therapeutic qualities for certain disease states.

Previous use of liposomes encapsulating glutathione has been limited byconcern that the combination would be adversely affected by the acidityand enzymes of the stomach. The preparation used in the presentinvention is able to deliver therapeutically active amounts ofglutathione to the system in spite of these concerns. The inventiondescribes the lipid encapsulation of the glutathione (reduced) into thelipid vesicle of liposomes and administered orally for the transmucosalabsorption into the nose, mouth, throat or gastrointestinal tractproviding the ability to conveniently supply therapeutically effectiveamounts of glutathione (reduce). The invention may also be administeredtopically for dermal and transdermal administration as well asintravenously.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1

Liposomal glutathione Drink or Spray 2500 mg per ounce

% w/w Deionized Water 71.9 Glycerin 15.00 Polysorbate-20 2.50 Lecithin1.50 Citrus Seed 0.50 Extract Potassium 0.10 Sorbate Glutathione 8.50(reduced)

Components lecithin, ethyl alcohol, cholesterol and glycerin werecommingled in a large volume flask and set aside for compounding(Alternatively, in all of the embodiments where the glutathione(reduced)percentage is 8.5, the glutathione (reduced percentage) can be loweredto 8.25 with 0.25% tocopherol acetate added).

In a separate beaker, water, hydroxy citric acid, glycerin, polysorbate20, glutathione were mixed and heated to 50 degrees C.

The water mixture was added to the lipid mixture while vigorously mixingwith a high speed, high shear homogenizing mixer at 750-1500 rpm for 30minutes.

The homogenizer was stopped and the solution was placed on a magneticplate, covered with parafilm and mixed with a magnetic stir bar untilcooled to room temperature. Citrus seed extract were added and thesolution was placed in appropriate dispenser for ingestion as a liquidor spray dispenser.

Analysis of the preparation under an optical light microscope withpolarized light at 400× magnification confirmed presence of bothmultilamellar lipid vesicles (MLV) and unilamellar lipid vesicles.

The preferred embodiment includes the variations of the amount ofglutathione to create less concentrated amounts of glutathione. Themethods of manufacture described in Keller et al, U.S. Pat No. 5,891,465are incorporated into this description.

A variation of the preferred embodiment of the invention is the additionof EDTA (ethylene diamine tetraacetic acid) 100 mg per ounce to beencapsulated in the liposome along with the glutathione.

Example 1A

Liposomal Glutathione Drink or Spray 2500 mg per Ounce or Form Suitablefor Encapsulation or Gel

% w/w Deionized Water 74.4 Glycerin 15.00 Lecithin 1.50 Citrus Seed 0.50Extract Potassium 0.10 Sorbate (optional spoilage retardant) Glutathione8.5 (reduced)

A lipid mixture having components lecithin, ethyl alcohol and glycerinwere commingled in a large volume flask and set aside for compounding.

In a separate beaker, a water mixture having water, glycerin,glutathione were mixed and heated to 50.degree. C.

The water mixture was added to the lipid mixture while vigorously mixingwith a high speed, high shear homogenizing mixer at 750-1500 rpm for 30minutes.

The homogenizer was stopped and the solution was placed on a magneticstirring plate, covered with parafilm and mixed with a magnetic stir baruntil cooled to room temperature. Normally, citrus seed extract would beadded. Normally, a spoilage retardant such as potassium sorbate or BHTwould be added. The solution would be placed in appropriate dispenserfor ingestion as a liquid or administration as a spray.

Analysis of the preparation under an optical light microscope withpolarized light at 400× magnification confirmed presence of bothmultilamellar lipid vesicles (MLV) and unilamellar lipid vesicles.

The preferred embodiment includes the variations of the amount ofglutathione to create less concentrated amounts of glutathione. Themethods of manufacture described in Keller et al U.S. Pat. No. 5,891,465are incorporated into this description.

Example 2

Liposomal Glutathione Drink or Spray 1000 mg per Ounce

With EDTA 1000 mg per ounce

% w/w Deionized Water 73.55 Glycerin 15.00 Polysorbate-20 2.50 Lecithin1.50 Citrus Seed Extract 0.50 Tocopherol Acetate 0.25 Potassium Sorbate0.10 Glutathione (reduced) 3.30 EDTA 3.30

Embodiment two of the invention includes the incorporation of the fluidliposome (such as that prepared in Example 1A) into a gelatin basedcapsule to improve the stability, provide a convenient dosage form, andassist in sustained release characteristics of the liposome. The presentembodiment relates to the use of glutathione in the reduced stateencapsulated into liposomes or formulated as a preliposome formulationand then put into a capsule. The capsule can be a soft gel capsulecapable of tolerating a certain amount of water, a two-piece capsulecapable of tolerating a certain amount of water or a two-piece capsulewhere the liposomes are preformed then dehydrated.

The liposome-capsule unit containing biologically encapsulated materialcan be taken in addition to orally, used for topical unit-of-useapplication, or other routes of application such as intra-occular,intranasal, rectal, or vaginal.

The composition of examples 1 and 2 may be utilized in the encapsulatedembodiment of this invention.

Gelatin capsules have a lower tolerance to water on their interior andexterior. The usual water tolerance for a soft gel capsule is 10% on theinterior. The concentration of water in a liposome formulation can rangefrom 60-90% water. An essential component of the present invention isthe formulation of a liposome with a relatively small amount of water,in the range of 5-10%. By making the liposome in a low aqueous system,the liposome is able to encapsulate the biologically active material andthe exposure of water to the inside lining of the capsule is limited.The concentration of water should not exceed that of the tolerance ofthe capsule for which it is intended. The preferred capsule for thisinvention is one that can tolerate water in the 1 5-20% range.

The method described by Keller et al, U.S. Pat. No. 6,726,924 areincorporated in this description.

Components are commingled and liposomes are made using the injectionmethod (Lasic, D., Liposomes, Elsevier, 88-90,1993). When liposomemixture cooled down 0.7 ml was drawn into a 1 ml insulin syringe andinjected into the open-end of a soft gelatin capsule then sealed withtweezers. The resulting capsule contains 10 mg CoQ10. Filling of gelcaps on a large scale is best with the rotary die method or others suchas the Norton capsule machine.

Example 3 Glutathione LipoCap Formulation

Ingredient Concentration (%) Sorbitan Oleate 2.0 Glutathione 89.8Purified Water 4.0 Potassium Sorbate 0.2 Polysorbate 20 2.0 Phospholipon90 (DPPC) 2.0

Components are commingled and liposomes are made using the injectionmethod (Lasic, D., Liposomes, Elsevier, 88-90, 1993). When liposomemixture cooled down 0.7 ml was drawn into a 1 ml insulin syringe andinjected into the open-end of a soft gelatin capsule then sealed withtweezers. The resulting one gram capsule contains 898 IU of Vitamin E.Large scale manufacturing methods for filling gel caps, such as therotary die process, are the preferred method for commercialapplications.

Embodiment number three of the present invention includes the creationof liposome suspension using a self-forming, thermodynamically stableliposomes formed upon the adding of a diacylglycerol-PEG lipid to anaqueous solution when the lipid has appropriate packing parameters andthe adding occurs above the melting temperature of the lipid. The methoddescribed by Keller et al, U.S. Pat. No. 6,610,322 is incorporated intothis description.

Most, if not all, known liposome suspensions are not thermodynamicallystable. Instead, the liposomes in known suspensions are kineticallytrapped into higher energy states by the energy used in their formation.Energy may be provided as heat, sonication, extrusion, orhomogenization. Since every high-energy state tries to lower its freeenergy, known liposome formulations experience problems withaggregation, fusion, sedimentation and leakage of liposome associatedmaterial. A thermodynamically stable liposome formulation which couldavoid some of these problems is therefore desirable.

The present embodiment prefers liposome suspensions which arethermodynamically stable at the temperature of formation. Theformulation of such suspensions is achieved by employing a compositionof lipids having several fundamental properties. First, the lipidcomposition must have packing parameters which allow the formation ofliposomes. Second, as part of the head group, the lipid should includepolyethyleneglycol (PEG) or any polymer of similar properties whichsterically stabilizes the liposomes in suspension. Third, the lipid musthave a melting temperature which allows it to be in liquid form whenmixed with an aqueous solution.

By employing lipid compositions having the desired fundamentalproperties, little or no energy need be added when mixing the lipid andan aqueous solution to form liposomes. When mixed with water, the lipidmolecules disperse and self assemble as the system settles into itsnatural low free energy state. Depending on the lipids used, the lowestfree energy state may include small unilamellar vesicle (SUV) liposomes,multilamellar vesicle (MLV) liposomes, or a combination of SUVs andMLVs.

In one aspect, the invention includes a method of preparing liposomes.The method comprises providing an aqueous solution; providing a lipidsolution, where the solution has a packing parameter measurement of Pa(P_(a) references the surface packing parameter) between about 0.84 and0.88, a P_(v) (P_(v) references the volume packing parameter) betweenabout 0.88 and 0.93, (See, D. D. Lasic, Liposomes, From Physics toApplications, Elsevier, p. 51 1993), and where at least one lipid in thesolution includes a polyethyleneglycol (PEG) chain; and combining thelipid solution and the aqueous solution. The PEG chain preferably has amolecular weight between about 300 Daltons and 5000 Daltons. Kineticenergy, such as shaking or vortexing, may be provided to the lipidsolution and the aqueous solution. The lipid solution may comprise asingle lipid. The lipid may comprise dioleolylglycerol-PEG-12, eitheralone or as one of the lipids in a mixture. The method may furthercomprise providing an active compound, in this case glutathione(reduced); and combining the active compound with the lipid solution andthe aqueous solution.

A variation of embodiment three is the combination of glutathione(reduced) and EDTA.

Additional variations of this embodiment of glutathione (reduced) andthe compounds claimed in this invention, including levodopa, carbidopa,Selenium, and EDTA are described in Keller et al, U.S. Pat. No.6,610,322.

Case Examples and Dosing

Liposomal Glutathione in Cystic Fibrosis

Case 1. MF aged 4 years has been diagnosed with cystic fibrosis and hasthe characteristic finding of elevated sweat chloride. She experiencesfrequent respiratory infections requiring antibiotic therapy and has achronic cough. Her mother described her as having decreased energy forplay, which restricted her physical activity. MF's red blood cell levelof glutathione was found to be 136 (normal range 200-400 micromole perL.) in December 2004.

Oral liposomal glutathione reduced was ingested in an amount thatprovided 300 mg glutathione per dose, with one dose per day for twoweeks. After two weeks ingesting the combination the individual'sglutathione level was found to be 570 micromole per L. During theinterval ingesting the oral liposomal glutathione reduced, theindividual was noted to have resolved the clinical symptoms of chroniccough, and to have more energy. Her mother described her as being ableto function normally after taking the oral liposomal glutathione. Thedose was adjusted down to 150 mg per dose, once a day, and theglutathione level reduced to 240 micromole per L., which has been usedfor a maintenance dose.

Case 2. Laura B

23 years old with CF manifesting with severe chronic lung disease, andchronic sinus congestion.

Her lung function had been unchanged at a very low level for 2 years.

Baseline RBC GSH was low

Baseline RBC GSH was low at 46 micromole per L. After 3 weeks of therapythe RBC GSH was 246 micromole per L. The normal range of RBC GSH is200-400 micromole per L.

Clinically, the patient noted a decrease in the amount of mucussecretions in both the sinuses and the lungs as well as an improvementin a cough, which had been chronic.

Case 3.

GF, an 18 month old girl with gastrointestinal manifestation of cysticfibrosis. In the first 12 months of life her growth pattern was normalwith her weight in the 50^(th) percentile. At the time of initialevaluation the child had fallen to the 25^(th) percentile for weight.Her glutathione blood levels were normal. The child was treated with theingestion of liposomal glutathione in a dose of 100 mg per thirty poundstwice a day.

After three months of ingesting the liposomal glutathione the child'sgrowth had returned to normal with her weight falling into the50-60^(th) percentile.

Dosing recommendation for the preferred embodiment of the invention, asdescribed in example 1.

Using oral liposomal glutathione 2500 mg per ounce.

Recommended Use

1 ounce is 5.56 teaspoons.

1 teaspoon of oral liposomal glutathione reduced contains approximately440 mg GSH.

Suggested dose depends on body weight. Recommended amounts are for dailyuse.

Gently stir liposomal glutathione into the liquid of your choice.

Refrigeration after opening is required to prevent deterioration.

Determine Daily Dose by Body Weight

Under 30 lbs: ¼ teaspoon=110 mg GSH

30-60 lbs: ½ teaspoon=220 mg GSH

60-90 lbs: ¾ teaspoon=330 mg GSH

90-120 lbs: 1 teaspoon=440 mg GSH

120-150 lbs: 1½ teaspoon=660 mg GSH

Over 150 lbs: 2 teaspoons=880 mg GSH

Parkinson's Disease:

A preferred application of the invention to Parkinson's disease is toinitially observe the response to glutathione therapy using theintravenous infusion of glutathione 1500 mg. One dose of the 1500 mg.glutathione intravenous is administered every 12 to 24 hours for a totalof 3 doses and the response to the therapy is observed. If there is apositive indication (improvement in the individual's symptoms ofParkinson's disease) for the continued use of glutathione, the presentinvention (liposomal glutathione) is utilized as an oral method ofmaintaining improvement of the Parkinson's symptoms.

Dosing Guide for Levodopa

Carbidopa/Levodopa: Each 10/100 tablet contains: carbidopa, 10 mg, andlevodopa, 100 mg.

Each 25/100 tablets contains: carbidopa, 25 mg, and levodopa, 100 mg.

Each 25/250 tablet contains: carbidopa, 25 mg, and levodopa, 250 mg.

Each sustained-release tablet contains: carbidopa, 50 mg, and levodopa,200 mg.

Case Example 3:

PP is 67-year-old woman with tremor affecting her hands, which isconsistent with Parkinson's disease. She relates being affected by thetremor to the point that she has difficulty closing buttons and writingher signature. She was placed on oral liposomal glutathione reduced, 600mg. twice a week. After three weeks of ingesting the invention, PP wasobserved to have significant reduction in her tremor. PP was able tofasten her buttons with more ease and was able to write her signaturewith less shaking.

Dosing Instructions for Liposomal Glutathione in Parkinson's Disease

The preferred initial therapy is the administration of 1.5 teaspoonsliposomal glutathione, which contains approximately 660 mg. ofglutathione twice a day for two weeks. If there is clinical improvementduring this time, the dose may be reduced to the level that maintainsthe good response on a continuing basis.

If there is no response at two weeks the therapy at the dose of 1.5teaspoons liposomal glutathione, which contains approximately 660 mg. ofglutathione twice a day until the conclusion of the time period, orclinical improvement has been achieved. If there is clinical improvementduring this time, the dose may be reduced to the level that maintainsthe good response on a continuing basis.

An alternative approach is to use the following:

Initial dose 1 ounce=2,500 mg

Repeat every 12 hours for a total or 4 doses.

Observe response and continue with dose that gives clinical responseusing

the following body weight indicator as dosing guide:

DETERMINE DAILY DOSE BY BODY WEIGHT AND Reduced glutathione RESPONSE TOTHERAPY: referred to as GSH  60-90 lbs: ¾ teaspoon 330 mg GSH  90-120lbs:   1 teaspoon 440 mg GSH 120-150 lbs: 1.5 teaspoon 660 mg GSH Over150 lbs:    2 teaspoons 880 mg GSH

The word “Selenium” means the chemical element selenium orpharmaceutically acceptable selenium-bearing compounds. Because Seleniumappears to facilitate biochemical cycles involving glutathione, thepurpose of Selenium is to be sure that sufficient selenium is present.

1. A liposome suspension suitable for oral administration, thesuspension comprising: 71.9% w/w deionized water, 8.5% w/w reducedglutathione, 15% w/w glycerin, 1.5% w/w lecithin, and 0.1% w/w potassiumsorbate.